Functional Organoids Research Articles

Biomaterial-Guided Organoid Engineering for Modeling Development and Diseases

Organoids are miniature models of organs to recapitulate spatiotemporal cellular organization and tissue functionality. The production of organoids has revolutionized the field of developmental biology, providing the possibility to study and guide human development and diseases in a dish. More recently, novel bio-material-based culture systems demonstrated the feasibility and versatility to engineer and produce the organoids in a consistent and reproducible manner. By engineering proper tissue micro-environment, functional organoids have been able to exhibit spatial-distinct tissue patterning and morphogenesis. This review will focus on enabling technologies in the field of organoid engineering, including chemical-defined hydrogels, micro-fabrication techniques and organoid-on-chip platforms, which facilitate the assembly of in vitro modeling of development and diseases.

A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids.

Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva-secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva-secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell-based approaches in three-dimensional (3D) scaffold-based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non-human-based substrates. Herein, saliva-secreting organoids/mini-glands were developed using a new scaffold/substrate-free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG-derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG-like organoids in well-established SG epithelial differentiation conditions for 7days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro-mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG-specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E-cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α-smooth muscle actin); and neuronal (β3-tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α-amylase activity assays showed functional organoids with SG-specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG-like mini-glands for SG secretory repair.

Tissue Specific Extracellular Matrix Extract Promotes Salivary Gland Organoid Formation with Human Salivary Progenitors

Hypofunction of the salivary glands (SGs) has several detrimental effects. Several approaches can be used to restore the function of damaged SGs, such as tissue engineering based on stem cells and biomaterials. Among these, three dimensional (3D) culture without biomaterials (termed organoids) can recapitulate the characteristics of the native organ in vivo. However, despite the substantial progress made, most organoids which are formed from stem cells or cell aggregates still lack a specific tissue niche. We hypothesized that tissue specific ECM proteins are required for formation and maintenance of functional organoids. Thus, we evaluate the effect of human tissue specific ECM extracts on the formation of functional SG organoids containing human SG epithelial and mesenchymal cells. We prepared human salivary gland ECM (hSG-ECM) with a growth factor-enriched supplement and the prepared hSG-ECM was introdeced in a uniform high-throughput 3D platform. First, we determined the optimum culture conditions; organoids comprising 5,000 cells at a mesenchymal: epithelial cell ratio of 1:9 exhibited a low frequency of apoptosis, high proliferation capacity, and a structure resembling that of SGs. Introduction of hSG-ECM extract with proper concentration resulted in maintenance of the stem cell niche and promotion of stem cell differentiation. Moreover, treatment of the organoids with decellularized supplements increased their sensitivity to neurotransmitters. In conclusion, we developed functional human SG organoids using salivary progenitors and ECM extract. The SG organoids have potential for SG tissue repair, drug development, and disease modeling.

Recapitulation of hepatitis B virus-host interactions in liver organoids from human induced pluripotent stem cells.

Therapies against hepatitis B virus (HBV) have improved in recent decades; however, the development of individualized treatments has been limited by the lack of individualized infection models. In this study, we used human induced pluripotent stem cell (hiPSC) to generate a functional liver organoid (LO) that inherited the genetic background of the donor, and evaluated its application in modeling HBV infection and exploring virus–host interactions. To establish a functional hiPSC-LO, we cultured hiPSC-derived endodermal, mesenchymal, and endothelial cells with a chemically defined medium in a three-dimensional microwell culture system. Based on cell-cell interactions, these cells could organize themselves and gradually differentiate into a functional organoid, which exhibited stronger hepatic functions than hiPSC derived hepatic like cell (HLC). Moreover, the functional LO demonstrated more susceptibility to HBV infection than hiPSC-HLC, and could maintain HBV propagation and produce infectious virus for a prolonged duration. Furthermore, we found that virus infection could cause hepatic dysfunction of hiPSC-LOs, with down-regulation of hepatic gene expression, induced release of early acute liver failure markers, and altered hepatic ultrastructure. Therefore, our study demonstrated that HBV infection in hiPSC-LOs could recapitulate virus life cycle and virus induced hepatic dysfunction, suggesting that hiPSC-LOs may provide a promising individualized infection model for the development of individualized treatment for hepatitis.

Bottom-up assembly of salivary gland microtissues for assessing myoepithelial cell function.

Myoepithelial cells are flat, stellate cells present in exocrine tissues including the salivary glands. While myoepithelial cells have been studied extensively in mammary and lacrimal gland tissues, less is known of the function of myoepithelial cells derived from human salivary glands. Several groups have isolated tumorigenic myoepithelial cells from cancer specimens, however, only one report has demonstrated isolation of normal human salivary myoepithelial cells needed for use in salivary gland tissue engineering applications. Establishing a functional organoid model consisting of myoepithelial and secretory acinar cells is therefore necessary for understanding the coordinated action of these two cell types in unidirectional fluid secretion. Here, we developed a bottom-up approach for generating salivary gland microtissues using primary human salivary myoepithelial cells (hSMECs) and stem/progenitor cells (hS/PCs) isolated from normal salivary gland tissues. Phenotypic characterization of isolated hSMECs confirmed that a myoepithelial cell phenotype consistent with that from other exocrine tissues was maintained over multiple passages of culture. Additionally, hSMECs secreted basement membrane proteins, expressed adrenergic and cholinergic neurotransmitter receptors, and released intracellular calcium [Ca2+i] in response to parasympathetic agonists. In a collagen I contractility assay, activation of contractile machinery was observed in isolated hSMECs treated with parasympathetic agonists. Recombination of hSMECs with assembled hS/PC spheroids in a microwell system was used to create microtissues resembling secretory complexes of the salivary gland. We conclude that the engineered salivary gland microtissue complexes provide a physiologically relevant model for both mechanistic studies and as a building block for the successful engineering of the salivary gland for restoration of salivary function in patients suffering from hyposalivation.

Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium.

In humans, the endometrium, the uterine mucosal lining, undergoes dynamic changes throughout the menstrual cycle and pregnancy. Despite the importance of the endometrium as the site of implantation and nutritional support for the conceptus, there are no long-term culture systems that recapitulate endometrial function in vitro. We adapted conditions used to establish human adult stem cell-derived organoid cultures to generate 3D cultures of normal and decidualised human endometrium. These organoids expand long-term, are genetically stable and differentiate following treatment with reproductive hormones. Single cells from both endometrium and decidua can generate a fully functional organoid. Transcript analysis confirmed great similarity between organoids and the primary tissue of origin. On exposure to pregnancy signals, endometrial organoids develop characteristics of early pregnancy. We also derived organoids from malignant endometrium, and so provide a foundation to study common diseases, such as endometriosis and endometrial cancer, as well as the physiology of early gestation.

Use of a Super-hydrophobic Microbioreactor to Generate and Boost Pancreatic Mini-organoids.

Cell remarkable ability to self-organize and rearrange in functional organoids has been greatly boosted by the recent advances in 3-D culture technologies and materials. This approach can be presently applied to model human organ development and function "in a dish" and to predict drug response in a patient specific fashion.Here we describe a protocol that allows for the derivation of functional pancreatic mini-organoids from skin biopsies. Cells are suspended in a drop of medium and encapsulated with hydrophobic polytetrafluoroethylene (PTFE) powder particles, to form microbioreactors defined as "Liquid Marbles," that stimulate cell coalescence and 3-D aggregation. The PTFE shell ensures an optimal gas exchange between the interior liquid and the surrounding environment. It also makes it possible to scale down experiments and work in smaller volumes and is therefore amenable for higher throughput applications.

In vitro generation of functional organoid structures resembling embryonic liver buds using differentiated, human cells

In vitro generation of functional organoid structures resembling embryonic liver buds using differentiated, human cells

The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells

Heterospheroids consisting of hepatocytes and bone marrow cells (BMCs) are formed by the mixed coculture of these cells and enhance the expression and maintenance of the liver-specific functions of hepatocytes. Not only the soluble factors derived from these cells, but also functional organoid (heterospheroid) formation, are considered to underlie this coculture effect. Therefore, in the present study, we aimed to clarify the mechanism of this co-culture effect. We performed hepatocyte monoculture with conditioned media prepared from hepatocyte cultures, BMC cultures and a coculture of hepatocytes and BMCs. When using any type of conditioned medium, no hepatocyte spheroids formed, and the hepatocytes formed a monolayer. In addition, an effect for these conditioned media was shown in terms of the albumin production and ammonia metabolism activities of the hepatocytes; conditioned medium from BMCs showed the strongest effect. The monocultured hepatocytes in the conditioned medium derived from BMCs showed equivalent albumin production and ammonia metabolism activities to the cocultured spheroids of hepatocytes and BMCs. Therefore, it was determined that the effect of the coculture of hepatocytes and BMCs was caused by soluble factors derived from BMCs.

Three-dimensional polarization sensitizes hepatocytes to Fas/CD95 apoptotic signalling

Maintenance of epithelial cell shape and polarity determines many vital cell functions, including the appropriate response to external stimuli. Murine hepatocytes cultured in a three-dimensional Matrigel matrix formed highly polarized organoids characterized by specific localization of an ERM (ezrin/radixin/moesin) protein, radixin, at microvillus-lined membrane domains. These apical domains surrounded a lumen and were bordered by tight junctions. The hepatocyte organoids were functional as judged by the high level of albumin secretion and accumulation of bilirubin. Stimulation of the Fas/CD95 death receptor, which is highly hepatotoxic in vivo, was a strong inducer of apoptosis in the polarized organoids. This was in sharp contrast to the monolayer hepatocyte cultures, which were protected from death by exacerbated NF-kappaB signalling following engagement of the death receptors. Thus, hepatocytes in polarized, functional organoids modulate an intracellular signal transduction pathway, allowing the recapitulation of their physiological response to an apoptotic stimulus.

Development of human prostate cancer models for chemoprevention and experimental therapeutics studies

The progression of human prostate cancer from histomorphologic to clinical expression often requires several decades. This study emphasizes the importance of developing relevant human prostate cancer models to study the molecular events leading to prostate cancer progression. These models will provide a rational basis for chemopreventive and treatment strategies to retard the progression of human prostate cancer from its localized to its metastatic state. In our laboratory, we have established the LNCaP progression and ARCaP models and the in vitro three-dimensional growth models involving prostate cancer and bone stroma to study the progression of prostate cancer. We propose that prostate cancer may progress from an androgen-dependent to an androgen-independent state. While existing as androgen-independent tumors (defined as tumors capable of growing in castrated hosts and secreting PSA in serum), prostate cancer may assume three different phenotypes as it progresses: androgen-independent while remaining androgen-responsive; androgen-independent and unresponsive to androgen stimulation; and androgen-independent but suppressed by androgen. It is conceivable that any androgen-independent human prostate cancer may contain variable proportions of cells that exhibit these three phenotypes. This concept may have important implications in determining strategies for chemopreventive and therapeutic trials. We have established three-dimensional growth models of prostate cancer cells either in collagen gel or microgravity-simulated growth conditions to form viable and functional organoids which contain prostate cancer epithelial cells admixed with prostate or bone stromal cells. These in vitro models combined with the in vivo models described above will enhance our understanding of the regulatory mechanism of prostate cancer growth and progression, and hence could improve efficiency in screening chemopreventive and therapeutic agents which alter the biologic behaviors of human prostate cancer.